| Assay Method Information | |
| | Fluorescence Polarization-Based Protein-Protein Interaction Inhibition |
| Description: | The instrument used in the FP experiment was the SpectraMax Multi-Mode Microplate Reader (Molecular Devices), and the excitation and emission wavelengths of the instrument were selected based on the corresponding fluorophore. The experiment was performed using a Corning 3676 384 well plate. The reaction system in the plate was 40 uL containing 10 uL of 4 nM FITC-9mer Nrf2 polypeptide fluorescent probe, 10 uL of 12 nM Keap1 Kelch domain protein solution and 20 uL of a corresponding concentration of an inhibitor. The positive control was 20 uL of 100 nM CPUY192002+10 uL probe+10 uL protein solution, the negative control was 10 uL probe+10 uL protein solution+20 uL HEPES buffer, and the blank control was 10 uL probe+30 uL HEPES buffer. Before the test, the system was mixed well and incubated for 30 minutes at room temperature. In this experiment, the probe fluorophore was fluorescein having an excitation wavelength of 485 nm and an emission wavelength of 535 nm. In this study, the fluorescence intensities in horizontal and vertical directions (F and F) were used to calculate the millipolarization value (mP), so as to reflect the changes of the polarized light. |
| Affinity data for this assay | |
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